A multitude of human defense systems have evolved to combat viral pathogens. This proposal aims to characterize a new mammalian antiviral immunity mechanism mediated by the RNA interference (RNAi) pathway in an adult mouse model. The innate and adaptive immunity mechanisms have been extensively characterized. The innate immune system is activated upon the detection of viral nucleic acids and is primarily regulated by type I interferons (IFN). The adaptive immune system is under the control of T and B lymphocytes that direct an antigen-specific and long-lasting antiviral immunity. In fungal, plant, insect and nematode hosts, viral dsRNA is processed by Dicer into small interfering RNAs (siRNAs) to direct a potent and specific antiviral defense by the conserved RNAi pathway. Recent studies have revealed production of abundant virus-derived siRNAs in mouse embryonic stem cells, differentiated hamster, monkey and human cells or suckling mice after infection with four positive- or negative-strand RNA viruses from three families. Moreover, three viruses have evolved distinct viral suppressors of RNAi (VSRs) with the same activity to inhibit the biogenesis of viral siRNAs and the RNA slicing activity of Argonaute2 is antiviral in cultured mouse and human cells. These findings lead us to hypothesize that there is a new mammalian antiviral immunity mechanism directed by the RNAi pathway. However, there has been much debate about whether the IFN response is inhibitory to antiviral RNAi. It also remains unknown whether mammalian viral siRNAs from an in vivo response can guide specific, Argonaute2-mediated cleavages of the targeted RNAs. Using Nodamura virus as a model for positive-strand RNA viruses, we will investigate the mechanisms in the induction and suppression of mammalian antiviral RNAi response in adult mice that are either active or defective in the IFN system. The proposed studies will reveal for the first time the properties, abundance and persistence of mammalian viral siRNAs in the infected adult mouse and show whether mammalian VSRs suppress antiviral RNAi and/or the IFN response during in vivo infection. We will characterize the antiviral activity of mammalian viral siRNAs made during active siRNA response of adult mice by two independent approaches. Furthermore, we will develop a conditional gene knockout system in adult mice for defining the in vivo antiviral function of the core RNAi pathway genes Dicer and Argonaute2, which has not been possible thus far in mammals due to their essential roles in animal development. Results from the proposed studies will provide both a mechanistic framework for the new mammalian antiviral response in vivo and an adult mouse model for antiviral RNAi, thereby facilitating investigations on the siRNA response induced by medically important RNA virus pathogens that are a major threat to human health.